Project Summary/Abstract G protein-coupled receptors (GPCRs) mediate most of our body's physiological responses to hormones, neurotransmitters and environmental stimulants. They are trans-membrane proteins that are expressed in every type of cell in the body where their function is to detect molecules outside the cell and transmit signals across the membrane to activate cellular responses. These proteins are involved in many diseases and present a wide range of opportunities as therapeutic targets in areas including diabetes, cancer, central nervous system disorders, inflammation, and pain. GPCRs have been and will remain among the most important targets of drug discovery in the pharmaceutical industry. In order to have sufficient and functional GPCR proteins for drug screening, an efficient heterologous protein expression system is required. The current heterologous expression systems using insect cells or mammalian cells typically result in low yields and require lengthy molecular biology efforts. E. coli is a very attractive protein expression system which offers flexibility in genetic manipulation, rapid growth, ease of scaling up and very low cost. However, current E. coli strains are limited in their ability to produce fully functional GPCR proteins. In this project, Primordial Genetics aims to develop a novel E. coli strain for producing functional GPCRs by a patented synthetic biology technology called Function Generator?. This technology has previously been applied to E. coli to generate functionality that cannot be created by classical genetic approaches. We propose to screen our combinatorial expression libraries to discover all the genes involved in improved expression of GPCRs. The expression of six well-known GPCRs will be validated by biochemical assays and function assays. At the end of this Phase I project, we will have demonstrated that for each target GPCR we have at least one FG gene that enables high-level expression of the soluble GPCR. Our Phase II project will comprise further strain development by stacking the trait genes to have a robust E. coli strain that is universally useful for GPCR production. The E. coli production host can be licensed to pharmaceutical companies and can be used to supply the market with GPCRs for functional screening. This endeavor not only bears great business prospects but also provides the scientific community with valuable R&D resources.